Department of Anatomy (Prof. T. Yamamoto) and Department of Physiology (Prof. K. Motokawa), Tohoku University School of Medicine, Sendai

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1 Tohoku J. exp. Med., 1966, 88, Electrical Excitability of Developing Cardiac Muscle in Chick Embryos Yoshifusa Shimizu and Kyoji Tasaki Department of Anatomy (Prof. T. Yamamoto) and Department of Physiology (Prof. K. Motokawa), Tohoku University School of Medicine, Sendai The resting and action potentials and the strength-duration relation to square current pulse stimulation were investigated on the chick embryo's heart from 2-day stage (7-somite stage) to near the hatching. From the embryos older than 3-day the resting and action potentials were recorded, and the magnitude of both potentials increased with the age. Before commencement of the spontaneous contraction (9-somite stage) electric stimulation failed to elicit contraction. After the 10-somite stage all-or-none contraction was produced by electric stimulation, and the strength-duration cruves were constructed at all stages by passing cathodal current to the cardiac muscle from 50ƒÊ micropipette. The rheobasic current was several tens of ƒêa at the earlier stages, decreasing rapidly for the first several days, and then fell gradually to less than 1 ƒêa at the later stages. The chronaxie was also found to shorten gradually from about 5 to 1 msec during the development. Since chronaxie is proportional to the membrane time constant, this characteristic change in chronaxie (membrane time constant) and rheobase (inverse of excitability) was suggested to correlate to structural change of the cell membrane. There have accumulated a large number of investigations concerning physio logical properties of developing cardiac muscles of the chick embryos (see Romanoff9). Among various physiological characteristics the electrical excitability as well as contractility and automaticity have been attracting considerable atten tion, and it was found that responsiveness to electricity applied externally develops concomitantly with appearance of the automatic contraction, and that the excitability to electric stimuli increases with the age. 3,5,7 In these experiments, a single or tetanizing current from an induction coil was applied to the spontaneously contracting heart and the effect of electric stimulation was investigated by observing the change in the heart rhythm. The induction coil, which was widely used in the classical studies in electrophysiology, is obviously inadequate for quantitative analysis of electrical properties of the excitable tissue. In the present study, a rectangular current pulse has been used to stimulate Received for publication, October 11,

2 50 Y. Shimizu and K. Tasaki the cardiac muscles taken from chick embryos of various developing stages and the strength-duration relation was investigated by taking an all-or-none contrac tion for determining the threshold. A current just sufficient to excite the heart muscle, the rheobase which is a reasonable index for excitability, was found to decrease rapidly first and then gradually with the age of embryo. Chronaxie, originally introduced to designate the time factor in excitation of the tissue, was obtained from the strength-duration curves and it was also found to take the similar time course to that of the excitability change. With intracellular microelectrode attemps were also made to record the resting and action potentials of the developing cardiac muscles, and it was shown that the magnitude of these potentials increased with the age. METHODS Materials. Chick embryos from the 7-somite stage (based on Lillie6) to 19-day were used. Except for the early stages of development at which the ventricular portion of the heart was not fully differentiated from the other parts, only the ventricle was extirpated and maintained in a glass container. The container was of a square shape made on a slide glass, and it was filled with the Gey's balanced salt solution which was frequently replaced with fresh solution during experiment. The temperature of the bathing fluid was kept at 30 Ž throughout the experiment by radiant heat from an infra-red lamp. Stimulation and recording. A stimulating electrode was a glass capillary filled with the Gey's solution. The tip of the stimulating pipette had been melted by electrically heated platinum wire under a microscope in order to obtain the exact internal diameter of 50ƒÊ. The smoothed tip of the stimulating electrode was impor tant to avoid a mechanical damage of the tissue when it was pressed against the heart muscle. A stimulating current was delivered from an electronic stimulator through a stimulus isolation unit against the indifferent g-agcil plate which was immersed in the bathing fluid. To pass a constant current a resistor (R) was connected in series with stimulating pipette, and the voltage drop across R was displayed on a cathode ray oscilloscope for accurate measurement of strength and duration of the stimulating current pulse. The voltage drop across R was also fed into an audioamplifier and to a loud speaker. The effect of electric stimulation, all-or-none contraction, was observed under a microscope. Intracellular recording from a single cardiac muscle fiber was achieved by glass capillary microelectrodes. For successful impalement of the electrode and for stable recording from contracting muscles, especially from the heart of early stages, it was necessary to use a long-shanked and flexible electrode which had a very fine tip. The resistance of the microelectrodes used was from 70 to 150 Meg ƒ and the tip diameter was 0.1 to 0.2ƒÊ (electronmicroscopic examination).

3 Excitability of Chick Embryo's Heart 51 The resting and action potentials were amplified by a negative capacitance cathode follower and a direct coupled amplifier, and displayed on a cathode ray oscilloscope. RESULTS 1. Resting membrane potential and action potential of the developing cardiac muscle The first appearance of the spontaneous contraction of the heart muscle was found, in the present study, at around the 10-somite stage. This is in agreement with the previous observations. 5,8,10 Before initiation of the spontaneous contraction, neither penetration of a single cell with microelectrode (indicated by sudden appearance of the resting membrane potential) nor stimulation of the muscle to contract were completely unsuccessful. Even after initiation of automaticity, from the 10-somite stage to about 3-day, penetration of a single cell with microelectrodes was extremely difficult. The first Fig. 1. Intracellular recordings from the excised ventricle of chick embryos. stable intracellular recording was made at the 3-day (18 stage). Such intracellular recordings were also made by Fingle, Woodbury and Hecht.4 As the incuba tion period progressed, penetration of a single cell appeared more easily. Fig. 1 shows the examples of the intracellular potentials. Both the resting and action potentials increased with the age. The relation between the incubation period and the magnitude of the intracellular potentials is further illustrated in Fig. 2. Even from the earliest stage at which successful recordings were made, the overshoot of the action potential was invariably seen. Low potential value at the early stages may reflect incomplete establishment of the cell membrane, but it must be also considered that at the early stages the fiber size was small and consequently microelectrode insertion caused more injury, resulting in attenua tion of intracellular potentials. Furthermore, the microelectrodes used in this study were extremely fine and their resistance was very high. Therefore the electrode tip potential would be expected to be high and varied one another, and the absolute value of the resting membrane potential could not be evaluated

4 52 Y. Shimizu and K. Tasaki Fig. 2. Resting and action potentials versus incubation period. Abscissa: age of embryo. Ordinate: resting ( œ) and action ( ü) potentials in mv. accurately.1 Another characteristic feature of the intracellular action potential of the developing cardiac muscle is the increase in the time derivative of the rising phase of the action potential (rate of rise). Although no systematic measurement was made, there was a general tendency for the rate of rise to decrease as the incubation period was longer. As to the duration of the action potential no definite relation was noted with the age. 2. Electrical responsiveness of the developing cardiac muscle Since stable intracellular recording from a single cardiac muscle fiber of the chick embryo was extremely difficult at the early stages, the excitability of the heart muscle of the early stages could not be investigated at the level of a single cell. On the other hand, all-or-none contraction of the whole preparation was found to be a useful indicator to determine the threshold of electrical stimula tion. Thus the electrical excitability was investigated by stimulating the heart monopolarly with the glass pipette of 50ƒÊ (see Methods). The stimulating electrode which had been filled with the Gey's solution was tightly pressed against the surface of the ventricle in order to minimize a leak of current flow to the lateral directions, so that a current would flow directly through the muscle fibers underneath the aperture of the pipette. Upon stimulating the ventricle of older than 10-somite stage at which the automaticity had already developed, the all-or-none contraction was invariably seen whenever the current was of sufficient strength. However, in the preparation

5 Excitability of Chick Embryo's Heart 58 of younger than the 9-somite stage, even the strongest obtainable current (3mA) failed to elicit any noticeable contraction (see also Discussion). Thus it is apparent that the automaticity and the electrical excitability develop simultaneously at about 10-somite stage. In this experiment the ventricle was artificially driven to contract by stimulating repetitively at frequency of slightly higher than the spontaneous rhythm of the preparation used. To determine the threshold for a fixed duration a sufficiently intense current to produce contraction was gradually decreased until the heart failed to respond to the stimulus. Just this intensity was taken as threshold. By this method of decreasing intensity, the threshold could be determined very accurately and remained unaltered throughout the experiment. Fig. 3. Strength-duration curves obtained from the excised ventricle of chick embryo. A: 2-day embyro (stage 11). B: 16-day embyro. Note decrease in rheobase and shortening of chronaxie in B as compared with those in A. On the other hand, the threshold determined with increasing intensity varied considerably from time to time, since the muscle was spontaneously contracting and the threshold intensity varied depending on the time after the previous contraction (relatively refractory period). To draw a strength-duration curve a cathodal current of various duration was applied and the threshold current was determined for each duration. At a fixed duration the threshold was always found much lower for cathodal current than for anodal one. Furthermore, the strength-duration curve of one preparation was almost identical at different region of the ventricle and easily replicated, so far as the size of the stimulating electrode was constant and the tip was tightly pressed upon the tissue. When the preparation was damaged locally during the operative procedure, there was considerable variation in threshold from one place to another. Such preparations were discarded. Fig. 3 shows two examples of the strength-duration relation obtained from the 2-day (A) and 16

6 54 Y. Shimizu and K. Pasaki day (B) embryos. It is noted that both the rheobase and chronaxie were markedly reduced at the later stage. Decrease in rheobase with the age is more clearly shown in Fig. 4. At the beginning of automaticity (a in Fig. 4) the rheobase was several tens of Đa and decreased very rapidly to the value of about 1 Đa within several days. During the rapid change in the rheobase and chronaxie, sensory fibers of the vagus nerve (c) and sympathetic elements (d) appear in the heart muscle. After this period the change in rheobase was more gradual reaching to the steady level. The change in the cardiac cycle (solid curve in Fig. 4) also appears to be approximately parallel to that of the rheobase. Fig. 4. Rheobase of the ventricle versus incubation period. Open circles show rheobase. Solid curve represents change of cardiac cycle, interval between successive contraction (calculated from the figure in Romanoff9). Arrow a: initiation of heart beat and appearance of myofilaments. b: formation of myofibrils. c: innervation by sensory fibers from the vagus nerve. d: appearance of sympathetic elements (Quoted from Romanoff9). From the strength-duration curve, chronaxie was obtained in each prepara tion. Gradual decrease in chronaxie with respect to the age of embryos is shown in Fig. 5. At the early stage chronaxie was about 5 msec long and

7 Excitability of Chick Embryo's Heart 55 progressively decreased down to less than 1 msec at the later stage. Fig. 5. Chronaxie versus incubation period. DISCUSSION The major findings in the present study are summarized as follows: 1. The electrical responsiveness of the heart appears simultaneously with automatic contraction. 2. The electrical excitability increases with the age. As to the irresponsiveness of the heart muscle to electric stimuli before commencement of the automatic heart beat, it might be argued that the stimula tion tested was not strong enough to produce contraction. In the present study, since stimulating current was limited by a series resistor and also micropipette, the maximally obtainable current intensity was 3 ma. A stronger current produced gas bubbles in the pipette which caused decrease in intensity. By eliminating the series resistor and pipette, much stronger current was tested with a silver wire insulated except the tip (100ƒÊ in diameter or larger sometimes), but no noticeable contraction or even local contraction was observed. Thus it is safe to conclude that the electrical irritability and automaticity are the concomitant phenomena in the developing heart muscle of the chick embryos. The chronaxie was defined, in the classical electrophysiology, as the least duration of a current pulse which excite the tissue at twice intensity of the rheobase, and the concept was considered to fill the need for an index of excitability of the tissue. This original concept of chronaxie was proved by many investigators to be only a rough measure of the time factor in excitation and only a poor correla tion was found between chronaxie and excitability.2 In the present study, however, a rather parallel relationship was found between chronaxie and excitability at least

8 56 Y. Shimizu and K. Tasaki during the developing period of the chick embryo's heart. For chronaxie is known to be directly proportional to the membrane time constant (chronaxie= Tm ~ln2, Tm: membrane time constant),11 the membrane time constant would be also expected to take the same time course, decreasing with the age. Direct measurement of Tm was not attempted with success at present because of several difficulties such as small size of the muscle fibers and high resistance of the microelectrode. Since the excitability and membrane time constant are the properties of the cell membrane, progressive change of these must be reflecting the structural change of the membrane itself, although development of interconnections among muscle fibers must be playing an important role in producing the all-or-none contraction of the whole muscle. Thus electronmicroscopic investigation of the heart muscle of developing chick embryos may provide the fundamental and useful knowledge for further understanding of the morphological correlates to electrical character istics of the cell membrane in general. Acknowledgment The authors express thanks to Prof. K. Motokawa, Department of Physiology and Prof. T. Yamamoto, Department of Anatomy, for their constant encouragement and support to this investigation. References 1) Adrian, R.H. The effect of internal and external potassium concentration on the membrane potential of frog muscle. J. Physiol. (Lond.), 1956, 133, ) Brazier, M.A.B. The electrical activity of the nervous system, Pitman Medical Publica tion Co., Ltd., London, ) Fano, G. Sullo svillupo della fuzione cardiaca neil embrione. Sperimentale, 1885, 1, ) Fingle, E., Woodbury, L.A. & Hecht, H.H. Effect of innervation and drugs upon direct membrane potentials of embryonic chick myocardium. J. Pharmacol. exp. Ther., 1952, 102, ) Johnstone, P.N. Studies on the physiological anatomy of the embryonic heart. Bull. Johns Hop. Hosp., 1925, 36, ) Lillie, F.R. Development of the chick, Hery Holt Co., Inc., New York, ) Olivo, O.M. Suil' inizio della fuzione contracttile del cuore e dei miotoni dell' embrione di polio in rapporto alla dolo differentione morfologica e strutturale. Arch. exp. Zellforsch., 1925, 1, ) Patten, B.M. & Kramer, T.C. The initiation of contraction in the embryonic chick heart. Amer. J. Anat., 1933, 53, ) Romanoff, A.L. The avian embryo, The Macmillan Co., New York, ) Sabin, F.R Quoted in Romanoff9. 11) Schafer, H. Uber die mathematisehen Grundlagen einer Spannungstheorie der elektrischen Nervenreizung. Pflugers Arch. ges. Physiol., 1936, 237,